N-methylimidazole may be used to synthesize oligonucleotides and/or phosphorothioate oligonucleotides. The synthesis generally has the following steps: (a) deblocking, (b) activation/coupling, (c) capping, and (d) oxidizing (in the case of oligonucleotides) or sulfurizing (in the case of phosphorothioate oligonucleotides). The cycle may be repeated sequentially depending on the number of bases to be coupled. The capping step is commonly carried out in the presence of a combination of N-methylimidazole and acetic anhydride. In some oligonucleotide syntheses employing N-methylimidazole, it has been observed that unwanted adducts may form. The adducts have been observed to add 85 daltons to the molecular weight of the oligonucleotides.
It has been determined that the formation of unwanted adducts occurs when the N-methylimidazole includes 1,3,5-trimethylhexahydro-1,3,5-triazine as an impurity. Specifically, 1,3,5-trimethylhexahydro-1,3,5-triazine has been found to react with the oligonucleotide to form unwanted adducts. Therefore, it would be beneficial to provide a method for removing 1,3,5-trimethylhexahydro-1,3,5-triazine from N-methylimidazole. Use of 1,3,5-trimethylhexahydro-1,3,5-triazine-free N-methylimidazole may provide for optimized synthesis of oligonucleotides and/or phosphorothioate oligonucleotides.
Accordingly, it is desirable to provide a method for removing 1,3,5-trimethylhexahydro-1,3,5-triazine from N-methylimidazole. Further, it is desirable to provide a method for forming oligonucleotides that includes the step of removing 1,3,5-trimethylhexahydro-1,3,5-triazine from N-methylimidazole before using the N-methylimidazole to cap unreacted deblocked nucleotide. Furthermore, other desirable features and characteristics will become apparent from the subsequent detailed description and the appended claims, taken in conjunction with the accompanying drawings and the foregoing technical field and background.